Gap junctional communication is essential for proper development and health of animal and human cardiac tissues. Gap junction channels establish communication by directly connecting the cytoplasm of adjacent cells. These channels are formed in vivo from several main proteins termed connexins that are coexpressed in embryonic and adult cardiac tissues. Since each connexin has its own perm-selectivity and gating properties, in vitro studies represent a strategy for understanding how the passage of endogenous metabolites through channels is regulated in vivo, and for determining how these connexins interact with one another to produce channels with new biophysical properties. We propose to continue our studies of gap junction channels formed by different cardiac connexins in order to further explore mechanisms that induce changes in permeability and selectivity. For Specific Aim #1, we will determine, through site-directed mutagenesis, how interactions between carboxyl tails of connexins alter the permeability of connexons, by focusing mainly on the phosphorylation of connexin43. In Specific Aim #2 we will use novel expression systems (funded during the previous cycle) to alter the ratio of individual connexins combined into the same connexon in order to determine the basic mechanism of selectivity to large molecules through these new channels, based on the size and charge of permeants, as well as changes in the charge of channels. For Specific Aim #3, we will employ novel tissue culture systems to determine how newly formed channels alter permeability to endogenous metabolites; these systems will have the advantage of obtaining data from cells containing regulated expression of connexins. Finally, in Specific Aim #4, we will consolidate information obtained during previous aims to focus on the physiological relevance of changes in permeability and cell-to-cell communication in the differentiation of tissues. Our model will be a cultured tumor cell line whose membrane potential and growth depends upon direct communication through gap junctions. Collectively, these experiments will be built on observations made during the previous funding cycle, and will further establish the relevance of heteromerization as a novel system to regulate, not only gating, but also perm-selectivity of gap junction channels.